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Abstract

Introduction

Brain-derived neurotrophic factor (BDNF) was first identified in the intervertebral
disc (IVD) when its molecular upregulation was observed in sections of nucleus pulposus
cultured under conditions of increased osmolarity. BDNF is now known to be involved
in a number of biologic functions, including regulation of differentiation/survival
of sensory neurons, regulation of nociceptive function and central pain modulation,
and modulation of inflammatory pain hypersensitivity. In addition, more recent investigations
show that BDNF can induce the recruitment of endothelial cells and the formation of
vascular structures. The objectives of the present study were to use immunocytochemistry
to determine the distribution of BDNF and its receptor (BDNF-tropomyosine receptor
kinase B) in the human IVD, and to test for gene expression of BDNF and its receptor
in cultured human annulus fibrosus cells.

Methods

We studied immunohistochemical localization of BDNF and its receptor in the human
annulus, quantified the percentage of outer annulus and inner annulus cells and nucleus
cells positive for BDNF immunolocalization, and studied the gene expression of BDNF
and its receptor using microarray analysis.

Conclusion

These findings provide data on the presence of BDNF and its receptor in the human
IVD at the translational level, and on the expression of BDNF and its receptor by
cultured human annulus cells. Our findings point to the need for further studies to
define the role of BDNF in the human IVD and to investigate regulatory events within
the disc that control the expression of BDNF and its receptor.

Introduction

Brain-derived neurotrophic factor (BDNF) was first identified in the intervertebral
disc (IVD) when its molecular upregulation was observed in sections of nucleus pulposus
cultured under conditions of increased osmolarity by Boyd and colleagues [1]. BDNF is now known to be involved in a number of biologic functions, including regulation
of differentiation/survival of sensory neurons, regulation of nociceptive function
and central pain modulation, and modulation of inflammatory pain hypersensitivity.

Nerves grow into tissues in response to signaling molecules called neurotrophins,
which are involved in the survival, differentiation, migration and outgrowth of neurons.
We now know that neurotrophins are expressed in non-neuronal tissues, including the
IVD [2-4]. Gigante and colleagues recently reported the presence of nerve growth factor (NGF)
mRNA, the high-affinity tyrosine kinase A receptor and the low-affinity p75 receptor
in rounded cells in the disc annulus and nucleus pulposus [5]. NGF and its receptors tyrosine kinase A and p75 are expressed not only in the developing
nervous system, but also in the mature adult nervous system – where they are particularly
important because of their association of primary nociceptive neurons. The recent
review by Mendell and colleagues points out that in adults NGF acts as an important
intermediate in pain, contributing to peripheral and central sensitization [6].

One other neurotrophin has previously been studied in the IVD. Abe and colleagues
have reported on the expression of NGF by human IVD cells in control disc tissue in vivo and in vitro in cells from control IVDs using immunocytochemistry [7]. NGF was found to be high in the outer annulus and in herniated disc tissue. Abe
and colleagues' work also demonstrated that the proinflammatory cytokines IL-1 and
TNFα had stimulatory effects on NGF. These authors suggested that such actions may
play a role in nerve sprouting into the IVD, and may be associated with discogenic
pain.

Ohtori and colleagues have reported on the presence of BDNF in dorsal root ganglion
neurons innervating lumbar discs in the rat [8]. Supraspinal BDNF-tyrosine kinase receptor B signaling is now known to represent
a mechanism underlying the development of persistent pain [9,10].

Another important role for BDNF involves more recent advances in our understanding
of BDNF function. The interesting studies by Kermani and colleagues have documented
the ability of BDNF to promote revascularization via endothelial cells and hematopoietic
progenitors [11]. These investigators suggest their work with skeletal muscle and heart indicates
that the signaling pathway involving BDNF and its receptor may also be active in modulation
of angiogenesis in specific organs in the adult. In light of the avascular status
of the adult IVD, this role merits future research.

Because of the significance of BDNF in the IVD, our objectives in the present work
were to use immunocytochemistry to determine the distribution of BDNF and its receptor
in the human IVD, and to test for gene expression of BDNF and its receptor in cultured
human annulus fibrosus cells.

Materials and methods

Clinical study population

The experimental study of human IVD specimens was approved prospectively by the authors'
Human Subjects Institutional Review Board (protocol number 08-04-09E). The need for
informed consent was waived (since surgical IVD tissue is routinely discarded at our
institution). Surgeries for disc degeneration are routine clinical practice in our
hospital. The Thompson grading system is used to score IVD degeneration over the spectrum
of stages from Thompson grade I (a healthy disc) to discs with advanced degeneration
(Thompson grade V) [12].

Patient specimens were derived from surgical disc procedures performed on individuals
with herniated discs and with degenerative disc disease. Surgical specimens were transported
to the laboratory (<30 min after surgical removal) in sterile tissue culture medium
and were placed in 10% neutral buffered formalin for no longer than 24 hours. Care
was taken to remove all granulation tissue and to sample only IVD tissue. Donor disc
specimens were obtained via the National Cancer Institute Cooperative Human Tissue
Network; they were shipped overnight to the laboratory in sterile tissue culture medium
and were processed as described below. Procurement of specimens from the Cooperative
Human Tissue Network was also approved via our Institutional Review Board. Specimens
were embedded in paraffin without decalcification.

Sand rat intervertebral disc tissue

Animal studies were performed following approval by the Institutional Animal Care
and Use Committee. Psammomys obesus, the sand rat, is used in our laboratory for studies of IVD degeneration. Colony
housing and animal diet descriptions have been published previously [13,14].

Spines from seven animals were used in the present study of the immunolocalization
of BDNF. The spines were removed immediately following animal euthanization, and the
IVDs were harvested, fixed in 10% neutral buffered formalin, decalcified, and embedded
in paraffin so that en face sections of the disc could be obtained. Sections were processed for immunohistochemical
localization of BDNF as described below.

BDNF immunohistochemistry performed on human intervertebral disc tissue and determination
of the percentage of positive cells

Paraffin sections were cut at 4 μm, collected on PLUS slides (Allegiance, McGaw Park,
IL, USA), dried at 60°C, deparaffinized in xylene (Allegiance) and rehydrated through
graded alcohols (AAPER, Shelbyville, KY, USA) to distilled water. Antigen retrieval
was performed using Dako Target Retrieval Solution, pH 6.0 (Dako, Carpenteria, CA,
USA) for 20 minutes at 95°C followed by cooling for 20 minutes. The remainder of the
procedure was performed using the Dako Autostainer Plus (Dako) automated stainer.
Endogenous peroxidase was blocked using 3% H2O2 (Humco, Texarcana, TX, USA). Slides were incubated for 1 hour with antihuman BDNF
(clone number 35928; R&D Systems, Minneapolis, MN, USA) at a 1:20 dilution. Mouse
IgG (Dako) was used as a negative control. The secondary antibody was Dako LSAB2 biotinylated
Link for HRP/AP (Dako) for 10 minutes followed by peroxidase-conjugated streptavidin
(Dako) for 10 minutes and DAB (Dako) for 5 minutes. Slides were removed from the stainer,
rinsed in water, counterstained with light green, dehydrated, cleared and mounted
with resinous mounting media.

Immunohistochemistry was performed as described above with the following exceptions.
The antibodies were biotinylated using the Dako ARK Kit (Dako). Sections were incubated
with streptavidin peroxidase (Dako ARK Kit) for 15 minutes at room temperature and
were then incubated in DAB (Dako) for 5 minutes. Rinses were performed between the
application of each reagent on the AutostainerPlus using Tris-buffered saline containing
Tween-20 (Dako). Slides were removed from the stainer, rinsed in water, counterstained
with 0.03% light green (Polysciences, Warrington, PA, USA), dehydrated cleared and
mounted with Cytoseal XYL (Richard-Allan Scientific, Kalamazoo, MI, USA).

BDNF-receptor immunolocalization

Embedding, sectioning and blocking endogenous peroxidase using 3% H2O2 were performed as described above. Slides were then incubated for 1 hour with rabbit
antibody to tyrosine kinase receptor B (BDNF receptor, batch number Rb68-010906-WS;
Biosensis, Flagstaff Hill, Australia) at a 1:500 dilution. Universal negative control
rabbit (Dako) was used as a negative control. The secondary antibody was Dako LSAB2
biotinylated Link for HRP/AP (Dako) for 10 minutes followed by peroxidase-conjugated
streptavidin (Dako) for 10 minutes and DAB (Dako) for 5 minutes. Slides were removed
from the stainer, rinsed in water, counterstained with light green, dehydrated, cleared
and mounted with resinous mounting media.

Human intervertebral disc cell culture

Annulus cells were grown in monolayer culture (seeded at 150,000 cells/well) on a
Lab-Tek® Chamber Slide™ (Nunc, Napierville, IL, USA) or in three dimensions in a collagen sponge
as previously described [15,16].

Gene expression studies

Human IVD tissue, annulus cells in a monolayer, and annulus cells in three-dimensional
culture were assayed for gene expression using the Affymetrix microarray system (Affymetrix,
Santa Clara, CA 95051, USA). Harvested cells were placed in extraction buffer from
the PicoPure RNA Isolation Kit (Arcturus, Mountainview, CA, USA), and total RNA was
extracted from the tissue using the PicoPure RNA Isolation Kit following the manufacturer's
instructions, were reverse transcribed to double-stranded cDNA, were subjected to
two rounds of transcription, and were hybridized to the DNA microarray in the Affymetrix
Fluidics Station 400 (Affymetrix, Santa Clara, CA 95051, USA). Affymetrix human U133
X3P arrays were used (Affymetrix, Santa Clara, CA 95051, USA). The GeneChip® Operating Software Affymetrix GeneChip Operating System (version 1.2; Affymetrix,
Santa Clara, CA 95051, USA) was used for determining gene expression levels.

Results

Immunohistochemical specimens used in the localization of BDNF and of the BDNF receptor
are described in Table 1. Thirty-one IVDs from 22 subjects were evaluated; three subjects were control donors,
and 19 subjects were patients undergoing disc surgery. The study design included discs
within each of the Thompson grade classifications. Since the majority of the surgical
specimens we obtain come from grade III and grade IV discs, there were fewer grade
I and grade V discs in the present study.

Table 1. Demographic features of specimens studied for immunocytochemical localization of brain-derived
neurotrophic factor and of its receptor

BDNF immunolocalization

Immunolocalization of BDNF was greatest in the outer annulus (Figure 1a,b). Localization of positive cells next to negative cells was also observed in the
outer annulus, the inner annulus and the nucleus (Figure 1c to 1e; Figure 1f is a negative control). Positive and negative cells were present within small clusters
(Figure 1e). Annulus cells encapsulated by the extracellular matrix also showed positive localization
(Figure 2).

The proportions of BDNF-positive cells were quantitatively determined for the outer
annulus, the inner annulus and the nucleus pulposus. The outer annulus contained an
average of 32.3 ± 2.7% (n = 22) (mean ± standard error of the mean) cells positive
for BDNF, a significantly greater level than that present in the inner annulus (8.1
± 1.5%, n = 6) or in the nucleus (10.4 ± 2.8%, n = 4) (P ≤ 0.01) (Figure 3). When data for the outer annulus were assessed based on the degenerative stage of
the IVD (Thompson grade), no significant relationship was present (Figure 4).

Sand rat lumbar IVDs sectioned en face showed a similar pattern of localization of BDNF. The greatest number of positive
cells was present in the outer annulus, with fewer positive cells as one viewed the
inner annulus (Figure 5).

BDNF-receptor immunolocalization

Localization patterns were similar for the BDNF receptor to that seen for BDNF. Both
positive cells and negative cells were present, with the greatest number of positive
cells present in the outer annulus (Figure 6). Positive cells were occasionally present in the nucleus in human specimens. Sand
rat lumbar IVDs showed a similar localization pattern, with a larger proportion of
positive cells in the outer annulus compared with the inner annulus or the nucleus
(Figure 7).

Gene expression of BDNF by annulus cells

The final portion of the present study looked at gene expression of BDNF by human
annulus cells cultured in monolayer and three dimensions. Annulus cells were all derived
from surgical patients. This experiment utilized one grade II disc (patient aged 45
years), five grade III discs (mean patient age 38.6 years), and four grade IV discs
(mean patient age 52.2 years). All cells were cultured for 10 days prior to cell harvest.
Positive expression levels were observed in all specimens for BDNF (gene identifier
NM_001709.1) per microarray analysis.

The BDNF gene expression levels correlated significantly and in a positive manner
with the Thompson disc grade for the cell cultures (P = 0.011, r = 0.72). When expression levels in grades II and grade III discs (the healthier discs
in this evaluation) were pooled and compared with mean expression levels in grade
IV discs, significantly higher expression was identified in the more degenerate IVDs
(P = 0.037; Figure 8).

Discussion

The present study presents novel immunocytochemical data demonstrating the presence
of BDNF and of BDNF receptor in the human outer annulus, inner annulus and nucleus
pulposus. Quantitative assessment of the proportion of cells positive for BDNF localization
showed significantly greater localization in the outer annulus compared with the inner
annulus or the nucleus pulposus (P ≤ 0.01). Quantitative analysis was not performed for the BDNF receptor, but qualitatively
its localization appeared to follow a similar pattern.

BDNF and the BDNF receptor were also found to be present in sand rat lumbar IVDs,
and the immunolocalization pattern was similar to that seen in human IVDs but with
more labeled cells present in the outer annulus. Inner annulus cell and nucleus cells
in the human IVD were found to occasionally show positive immunolocalization, but
at a significantly lower proportion. Although there was minimal background present
in the animal tissues, it must be remembered that this portion of our study utilized
antihuman antibodies, no biochemical analysis of the cross-reactivity of the sand
rat substrates was carried out, and a danger of some false-positive reactivity with
proteins in the sand rat IVD remains a concern that at present cannot be discounted.

Neurotrophins are beginning to be viewed as important agents in the IVD. Aoki and
colleagues have suggested that NGF-dependent neurons may be the nerve population responsible
for discogenic pain based on findings from studies of disc degeneration in rats [17,18]. A study of human discs from subjects with low back pain performed by Freemont and
colleagues identified a positive relationship between ingrowth of nonmyelinated, nociceptive
nerves into the IVD and the production of nerve growth factor by nearby ingrowing
blood vessels [19].

Studies have recently shown production of several neurotrophins by IVD cells, but
the mechanisms that control their expression, and the effects these factors have on
disc cells, are poorly understood. Gigante and colleagues recently reported the presence
of NGF mRNA and the high-affinity tyrosine kinase A receptor and the low-affinity
p75 receptor in the rounded cells in the disc annulus and nucleus pulposus [5]. The p75 receptor is of special interest because it also has the ability to bind
to BDNF [20].

The work presented here adds to, and expands, our understanding of the distribution
of BDNF and its receptor in the human IVD. In addition to NGF, BDNF has recognized
neuroprotective effects [21]. There is also evidence that BDNF can be considered a neurotransmitter and may have
a role in nociceptive transmission [22]. Work by Thompson and colleagues suggests that BDNF may be released from primary
sensory nociceptors with activity such as that involved with persistent pain states
and inflammatory conditions [9]. It is well recognized that differential regulation of neurotrophin expression occurs
in inflammatory states, such as in the airway, where NGF, BDNF and neurotrophin 3
have been shown to be elevated during inflammation [23]. Increased proinflammatory cytokines are now well recognized during IVD degeneration
[24-27], providing support for the hypothesis that BDNF in vivo may be produced in response to inflammatory cytokines during IVD degeneration. Clearly
the relationship of BDNF to inflammatory cytokines in the IVD merits future research
attention.

The recent studies of by Kermani and colleagues regarding the ability of BDNF to promote
revascularization via endothelial cells and hematopoietic progenitors are currently
unexplored in the IVD [11]. These investigators suggest that the signaling pathway involving BDNF and its receptor
may function in modulation of angiogenesis in specific organs in the adult. Since
the adult disc is avascular, the potential of BDNF to influence angiogenesis in the
mature disc is intriguing.

Finally, the initial observations by Boyd and colleagues that showed the upregulation
of BDNF by application of increased osmolarity remains intriguing [1], and suggests the potential importance of the unexplored relationship between mechanoregulation
and BDNF in the human IVD.

Conclusion

The data presented here document the presence of BDNF and its receptor in the human
IVD at the translational level, and also show the expression of BDNF and its receptor
by cultured human annulus cells. Quantitative assessment of the proportion of cells
positive for BDNF localization showed that the outer annulus contained a significantly
larger proportion of positive cells compared with the inner annulus or the nucleus
(P ≤ 0.01). Our findings point to the need for further studies to define the role of
BDNF in the human IVD and to investigate regulatory events, especially those related
to mechanoregulation, which regulate the expression of BDNF and its receptor in the
human IVD.

Abbreviations

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

HEG conceived of the study and participated in study design with ENH, analyzed the
data, and prepared and revised the manuscript. JAI, GH and NZ performed laboratory
work and analysis. HJN performed statistical analyses and assisted with manuscript
preparation. All authors approved the final manuscript.

Acknowledgements

The authors wish to thank the Brooks Center for Back Pain Research for its support.